Human prostatic specific reductase

ABSTRACT

A human prostatic specific reductase polypeptide and polynucleotides encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polynucleotides as a diagnostic marker for prostate cancer and as an agent to determine if the prostate cancer has metastasized. Also disclosed are antibodies specific to the prostatic specific reductase polypeptide which may be used to target prostate cancer cells and be used as part of a prostate cancer vaccine. Methods of screening for agonists and antagonists for the polypeptide and therapeutic uses of the antagonists are also disclosed.

This is a Divisional of application Ser. No. 08/464,400, filed Jun. 5,1995, now allowed as U.S. Pat. No. 5,786,204 which is aContinuation-In-Part of PCT/US95/01827, filed Jan. 20, 1995.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, and the use of such polynucleotides andpolypeptides as part of a diagnostic assay for detecting the presence ofprostate cancer and prostate cancer metastases. The polynucleotides andpolypeptides of the present invention are human prostatic specificreductase, and are sometimes hereinafter referred to as “PSR”.

Carcinoma of the prostate has long been regarded as an unpredictabledisorder which makes sound therapeutic decisions in evaluating theresults of different types of treatment very difficult. Prostate canceris unique among the potentially lethal human malignancies in that thereis a wide discrepancy between the high prevalence of histologic changesrecognizable as cancer and the much lower prevalence of clinicaldisease.

The concept that adenocarcinoma of the prostate exists in a latent and aclinical form is supported by epidemiologic, pathologic and clinicalevidence. Although these divergent manifestations of prostate cancerhave come in architectural and cytologic features, they can bedistinguished from each other to some degree by differences in certainpathologic features, such as the volume, grade, and invasiveness of thelesion.

Prostate cancer has become the most common cancer among American men,and only lung cancer is responsible for more cancer deaths (Boring, C.C., Cancer Statistics, 41:19-36 (1991)). The age specific mortality ratehas slowly increased over the past 50 years and in black American men isnearly double the rate found in white men (Carter, H. B., Prostate,16:39-48 (1990)). Prostate cancer is responsible for nearly threepercent of all deaths in men over the age of 55 years (Seidman, H., etal., Probabilities of Eventually Developing or Dying of Cancer-UnitedStates, 35:36-56 (1985)). Since the incidence of prostate cancerincreases more rapidly with age than any other cancer, and the averageage of American men is rising, the number of patients with prostatecancer is expected to increase dramatically over the next decade.

Approximately 30% of men with prostate cancer have distant metastases atthe time of diagnosis (Schmidt, J. D., et al., J. Urol., 136:416-421(1986)). Despite the impressive symptomatic response of metastases tohormonal manipulation (androgen deprivation), the survival rate forthese patients is dismal: the median duration of survival is less thanthree years (Eyar, D. P., Urologic Pathology: The Prostate,Philadelphia, Pa., Lea and Febiger, 241-267 (1977)). By five years, over75% and by ten years, more than 90% of these patients die of theircancer rather than with it (Silverberg, E., Cancer, 60:692-717 (1987)(Suppl.)).

The problem with prostate cancer is that many forms of prostate cancerare latent, in other words, are difficult to detect. Approximately 30%of the men over the age of 50 years who have no clinical evidence ofprostate cancer harbor foci of cancer within the prostate (McNeal, J.E., et al., Lancet January, 11:60-63 (1986)). This remarkably highprevalence of prostate cancer at autopsy, seen in no other organ, makesit the most common malignancy in human beings (Dhom, G., J. Cancer Res.Clin. Oncol., 106:210-218 (1983)). There is strong support for theconcept of multi-step process in the pathogenesis of prostate cancer inwhich latent cancers progress through some but not all of the stepsnecessary for full malignant expression (Utter, H. B., et al., J. Urol.,143:742-746 (1990).

There are a variety of techniques for early detection andcharacteristics of prostate cancers, however, none of them are devoid ofany problems. Prostate cancer is a notoriously silent disease with fewearly symptoms. Symptoms associated with bladder outlet obstruction arecommonly present in men over the age of 50 years and are often ascribedto benign prostatic hyperplasia (BPH).

Digital rectal examination (DRE) traditionally has been considered themost accurate test for the detection of prostate cancer. DRE has beendemonstrated to be more sensitive, more specific, and to have a greaterefficiency than a variety of laboratory tests available, however, few ofthese laboratory tests are still in clinical use today (Guinan, P., etal., N. Engl. J. Med., 303:499-503 (1980)). DRE detects cancerrelatively late, and there is only a weak correlation between the sizeof the cancer estimated by DRE and the actual volume of cancer present.The most serious limitation of DRE is its lack of sensitivity(false-negative results). For example, approximately 10% to 20% oftransurethral resections performed for benign prostatic hypertrophy inpatients with no palpable abnormalities suggestive of cancer uncover anincidental cancer of the prostate. DRE detected only 12 of 22 cancersfound in a screening study, while transrectal ultrasonography (TRUS)found 20 (Lee, F., et al., Radiology, 168:389-394 (1988)). Thus, DRE isrelatively insensitive and nonspecific. Cancers detected by palpationare relatively large, late in their development and no longer curable,and some are very small, such that they are clinically unimportantcancers.

Patients having prostate cancer have an elevated prostate-specificantigen level. Cancer was detected in 26% of the men with a PSA level of4.0 to 10.0 ng/ml. Serum PSA levels have been shown to correlategenerally with the volume, clinical stage, and pathologic stage ofprostate cancer, although there is a wide range of PSA values associatedwith any given volume or stage (Hudson, M. A., J. Urol., 142:1011-1017(1989)). PSA, however, is not predictive of the features of the cancerin the individual patient. If the level of PSA is greater than 10.0ng/ml, 57% to 92% of the patients will have locally advanced cancer.Therefore, while more specific, using a PSA level higher than 10 ng/mlmay not offer an effective technique for early detection. There areother theoretical limitations to the use of this serum marker for earlydetection. A normal serum PSA level does not exclude the diagnosis ofcancer. False-negative results are common, and a third of men treatedwith radioprostatectomy for prostate cancer have a normal serum PSAlevel. False-positive results are also common since PSA levels are oftenelevated in men with common benign conditions, such as BPH orprostatitis. In summary, PSA levels have proved to be extremely usefulin the early detection of prostate cancer, especially when combined withDRE or TRUS. A PSA level detection, however, must be used in combinationwith DRE or TRUS in order to be sure that what is present is cancer andnot BPH or prostatitis.

The introduction of TRUS has provided physicians with an effective wayto see the internal anatomy and pathology of the prostate gland. TRUShas been used to screen for prostate cancer in several large series andhas consistently been shown to increase detection when compared withDRE. TRUS is performed by taking a sonograph of the pelvic area andperhaps the most important use of TRUS in the early detection ofprostate cancer is as a guide for directed needle biopsies of theprostate (Lee, F., et al., Radiology, 170:609-615 (1989)).

In accordance with one aspect of the present invention there is provideda method of and products for diagnosing prostate cancer metastases bydetermining the presence of specific nucleic acid sequences in a samplederived from a host.

In accordance with another aspect of the present invention, there isprovided a method of and products for diagnosing a prostate disorder bydetermining an altered level of PSR protein in a biological samplederived from a host, whereby an elevated level of PSR protein indicatesa prostate disorder diagnosis.

In accordance with another aspect of the present invention, there isalso provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to the prostatic specificreductase genes and polypeptides of the present invention.

In accordance with a further aspect of the present invention, there areprovided novel polypeptides which are prostatic specific reductasepolypeptides, as well as biologically active and diagnostically ortherapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human PSR proteins,including mRNAs, DNAS, cDNAs, genomic DNAs, as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogs,and derivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing human prostatic specific reductasenucleic acid sequences, under conditions promoting expression of saidproteins and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, thereare provided antibodies specific to such polypeptides.

In accordance with another aspect of the present invention, there areprovided processes for using the PSR polypeptides of the presentinvention to screen for compounds, for example, antagonists and/oragonists and antibodies which interact with the polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof prostate cancer.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1A and 1B, collectively show the cDNA sequence and thecorresponding deduced amino acid sequence of the PSR polypeptide. Thestandard one-letter abbreviations for amino acids is used.

FIG. 2A, 2B 2C and 2D collectively are the homology of PSR to otherreductases.

The boxed amino acids are those which correspond exactly between thepolypeptides.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention there is provideda diagnostic assay for detecting micrometastases of prostate cancer in ahost. While applicant does not wish to limit the reasoning of thepresent invention to any specific scientific theory, it is believed thatthe presence of mRNA encoding PSR in cells of the host, other than thosederived from the prostate, is indicative of prostate cancer metastases.This is true because, while the PSR genes are found in all cells of thebody, their transcription to mRNA and expression of the encodedpolypeptide is limited to the prostate in normal individuals. However,if a prostate cancer is present, prostate cancer cells migrate from thecancer to other cells, such that these other cells are now activelytranscribing and expressing the PSR genes and the mRNA is present. It isthe detection of this mRNA or expressed protein in cells, other thanthose derived from the prostate, which is indicative of metastases ofprostate cancer.

In such a diagnostic assay, a nucleic acid sequence in a sample derivedfrom a tissue other than the prostate is amplified and detected. Thesample contains a nucleic acid or a mixture of nucleic acids, at leastone of which is suspected of containing the sequence coding for PSRpolypeptide. Thus, for example, in a form of an assay for determiningthe presence of a specific mRNA in cells, initially RNA is isolated fromthe cells.

There are numerous methods, which are well known in the art, fordetecting the presence of a specific nucleic acid sequence in a sampleobtained from cells, such as from blood, urine, saliva, tissue biopsy,and autopsy material. The use of such assays for detecting mRNAtranscribed from the PSR gene in a sample obtained from cells derivedfrom other than the prostate is sell within the scope of those skilledin the art from the teachings herein.

The isolation of mRNA comprises isolating total cellular RNA bydisrupting a cell and performing differential centrifugation. Once thetotal RNA is isolated, mRNA is isolated by making use of the adeninenucleotide residues known to those skilled in the art as a poly(A) tailfound on virtually every eukaryotic mRNA molecule at the 3′ end thereof.Oligonucleotides composed of only deoxythymidine [oligo(dT)] are linkedto cellulose and the oligo(dT)—cellulose packed into small columns. Whena preparation of total cellular RNA is passed through such a column, themRNA molecules bind to the oligo(dT) by the poly(A)tails while the restof the RNA flows through the column. The bound mRNAs are then elutedfrom the column and collected.

One example of detecting mRNA encoding for a specific protein, forexample PSR, comprises screening the collected mRNAs with gene specificoligonucleotide probes which have been custom designed to hybridize tothe mRNA to be detected. Probing technology is well known in the art andit is appreciated that the size of the probes can vary widely but it ispreferred that the probe be at least 15 nucleotides in length. It isalso appreciated that such probes can be and are preferably labeled withan analytically detectable reagent to facilitate identification of theprobe. Useful reagents include but are not limited to radioactivity,fluorescent dyes or enzymes capable of catalyzing the formation of adetectable product.

Another method for detecting a specific mRNA sequence utilizes thepolymerase chain reaction (PCR) in conjunction with reversetranscriptase. PCR is a very powerful method for the specificamplification of DNA or RNA stretches (Saiki et al., Nature, 234:163-166(1986)). One application of this technology is in nucleic acid probetechnology to bring up nucleic acid sequences present in low copynumbers to a detectable level. Numerous diagnostic and scientificapplications of this method have been described by H. A. Erlich (ed.) inPCR Technology-Principles and Applications for DNA Amplification,Stockton Press, USA, 1989, and by M. A. Inis (ed.) in PCR Protocols,Academic Press, San Diego, USA, 1990.

RT-PRC is a combination of PCR with an enzyme called reversetranscriptase. Reverse transcriptase is an enzyme which produces cDNAmolecules from corresponding mRNA molecules. This is important since PCRamplifies nucleic acid molecules, particularly DNA, and this DNA may beproduced from the mRNA separated from the body sample derived from thehost. An example of an RT-PCR diagnostic assay involves removing asample from a tissue of a host. Such a sample will be from a tissue,other than the prostate, extracting total RNA from the sample,performing PSR RT-PCR of total RNA and electrophoresing on an agarosegel the PCR products. The oligonucleotide primers used for RT-PCR arebetween 16 and 50 nucleotide bases in length and preferably between 16and 30 nucleotide bases in length. Any segment of the PSR mRNA sequencemay be used to generate the oligonucleotides. In this manner, once thesequences aer amplified using PCR, genomic DNA may be distinguished fromPSR mRNA since different size bands will appear after electrophoresis ona 1.2% agarose gel. The presence of genomic DNA, an approximately 1.2 kbband, is a reading which is a negative indication concerning metastases.However, a much smaller band indicates mRNA is present which means thePSR gene is being actively transcribed which in turn indicates prostatecells are circulating in the blood and possibly metastisizing.

Another example for detecting a specific RNA in a sample involvesgenerating a cDNA molecule which corresponds to the mRNA to be detectedby the use of reverse transcriptase and, thereafter, cloning the cDNAmolecule into a vector to prepare a library. Such a method involvestransforming bacterial cells with the plasmid and spreading the cellsonto the surface on an agar plate that contains nutrients for growth andappropriate antibiotics for selection. In this manner all of the mRNAsfrom the collected fraction are transformed into the bacteria and platedout into individual colonies.

The mRNA may be detected from the library in a variety of methods. Forexample nucleic acid probes may be used to locate clones carrying adesired cDNA sequence. In such a method a replica of the library isprepared on nitrocellulose filters. This process transfers a portion ofeach colony to the nitrocellulose. Screening is carried out byincubating these nitrocellulose replicas with a nucleic acid probe withan antibody which is specific to the cDNA corresponding to the mRNA tobe detected.

The presence of mRNA transcribed from PSR in cells derived from otherthan the prostate may also be determined by use of an assay whichdetects the expression product of such gene. Thus, for example, such anassay involves producing cDNA from the mRNA contained in the sample andthen determining the presence of a specific mRNA by detecting theexpression product of the cDNA produced therefrom.

In such a method cDNAs are identified by searching for their geneproducts in bacteria after cloning the cDNA into appropriatelyconstructed plasmids termed expression vectors. cDNAs are inserted intothese vectors within regions that promote their expression in E. coli.Regulatable bacterial promoters are used. Proteins may be expressed asfusion proteins in which amino acids from a prokaryotic protein areincorporated at one end of the eukaryotic protein. Fusion proteins aremore stable than corresponding eukaryotic protein in bacteria and aretherefore produced at higher levels.

Another method to detect the mRNA sequence is to locate clones thatexpress a desired protein and assay for the function of the protein, forexample PSR. Radioactively labelled substrate for PSR may be used as aprobe to identify clones expressing proteins that are able to associatewith the substrate in vitro.

The cloned genes may also be identified by functional assay ineukaryotic cells. This technique allows direct physical selection ofcells expressing the cDNA of interest, and this cDNA can be recovereddirectly from the cells. Mammalian genes can also be isolated by geneticselection for their function in recipient cells.

The presence of active transcription from the PSR gene to producecorresponding mRNA in cells other than the prostate is an indication ofthe presence of a prostate cancer which has metastasized, since prostatecancer cells are migrating from the prostate into the generalcirculation. Accordingly, this phenomenon may have important clinicalimplications since the method of treating a localized, as opposed to ametastasized, tumor is entirely different. The presence of PSR mRNA inthe peripheral venous blood is an indication of metastases.

The assays described above may also be used to test whether bone marrowpreserved before chemotherapy is contaminated with micrometastases of aprostate cancer cell. In the assay, blood cells from the bone marrow areisolated and treated as described above, this method allows one todetermine whether preserved bone marrow is still viable fortransplantation after chemotherapy.

This invention is also related to use of the PSR genes as a diagnostic.For example, some diseases result form inherited defective genes. Amutation in the genes at the DNA level may be detected by a variety oftechniques. Nucleic acids used for diagnosis (genomic DNA, mRNA, etc.)may be obtained from a patient's cells, other than from the prostate,such as from blood, urine, saliva, tissue biopsy and autopsy material.The genomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR prior to analysis. RNA or cDNA may also beused for the same purpose. As an example, PCR primers complementary tothe nucleic acid of the instant invention can be used to identify andanalyze PSR gene mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabelled PSR gene RNA or, alternatively,radiolabelled antisense DNA sequences. Perfectly matched sequences canbe distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between the reference gene and “mutants” may berevealed by the direct DNA sequencing method. In addition, cloned DNAsegments may be used as probes to detect specific DNA segments. Thesensitivity of this method is greatly enhanced when combined with PCR.For example, a sequencing primer is used with double-stranded PCRproduct or a single-stranded template molecule generated by a modifiedPCR. The sequence determination is performed by conventional procedureswith radiolabelled nucleotides or by automatic sequencing procedureswith fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments andgels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high-resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g.,Myers, et al., Science, 230:1242 (1985)). In addition, sequencealterations, in particular small deletions, may be detected as changesin the migration pattern of DNA.

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and Si protection or the chemicalcleavage method (e.g., Cotton, et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of the specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting.

In accordance with another aspect of the present invention, there isprovided a method of diagnosing a disorder of the prostate, for examplecancer, by determining atypical levels of PSR products in a biologicalsample, derived from a tissue other than the prostate. Assays used todetect levels of PSR proteins in a sample derived from a host arewell-known to those with skill in the art and include radioimmunoassays,competitive-binding assays, western blot analysis, ELISA assays and“sandwich” assays. A biological sample may include, but is not limitedto, tissue extracts, cell samples or biological fluids, however, abiological sample specifically does not include tissue or cells of theprostate. An ELISA assay (Coligan, et al., Current Protocols inImmunology, 1(2), Chapter 6, 1991) initially comprises preparing anantibody specific to PSR proteins, preferably a monoclonal antibody. Inaddition, a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g., a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like BSA. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any PSR proteins attached to the polystyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody linked to horseradish peroxidase is now placed in the dishresulting in binding of the reporter antibody to any monoclonal antibodybound to PSR proteins. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount of PSRproteins present in a given volume of patient sample when comparedagainst a standard curve.

A competition assay may be employed where an antibody specific to PSRproteins are attached to a solid support and labeled PSR proteins and asample derived from the host are passed over the solid support and theamount of label detected, for example, by liquid scintillationchromatography, can be correlated to a quantity of PSR proteins in thesample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assay,PSR proteins are passed over a solid support and bind to antibodyattached to the solid support. A second antibody is then bound to thePSR proteins. A third antibody which is labeled and is specific to thesecond antibody, is then passed over the solid support and binds to thesecond antibody and an amount can then be quantified.

In alternative methods, labeled antibodies to PSR proteins are used. Ina one-step assay, the target molecule, if it is present, is immobilizedand incubated with a labeled antibody. The labeled antibody binds to theimmobilized target molecule. After washing to remove the unboundmolecules, the sample is assayed for the presence of the label. In atwo-step assay, immobilized target molecule is incubated with anunlabeled antibody. The target molecule-labeled antibody complex, ifpresent, is then bound to a second, labeled antibody that is specificfor the unlabeled antibody. The sample is washed and assayed for thepresence of the label.

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of marker is readilydeterminable to one skilled in the art. These labeled antibodies may beused in immunoassays as well as in histological applications to detectthe presence of the proteins. The labeled antibodies may be polyclonalor monoclonal.

Such antibodies specific to PSR, for example, anti-idiotypic antibodies,can be used as a prostate cancer vaccine since the antibodies preventthe action of PSR by binding tightly thereto, and, therefore, prevent oreliminate the viability of prostate cancer cells.

The antibodies may also be used to target prostate cancer cells, forexample, in a method of homing interaction agents which, when contactingprostate cancer cells, destroy them. This is true since the antibodiesare specific for PSR which is primarily expressed in the prostate, and alinking of the interaction agent to the antibody would cause theinteraction agent to be carried directly to the prostate.

Antibodies of this type may also be used to do in vivo imaging, forexample, by labeling the antibodies to facilitate scanning of the pelvicarea and the prostate. One method for imaging comprises contacting anytumor cells of the prostate to be imaged with an anti-PSR antibodylabeled with a detectable marker. The method is performed underconditions such that the labeled antibody binds to the PSR. In aspecific example, the antibodies interact with the prostate, forexample, prostate cancer cells, and fluoresce upon such contact suchthat imaging and visibility of the prostate is enhanced to allow adetermination of the diseased or non-diseased state of the prostate.

To determine if the amount of PSR polypeptide is elevated, the methodsdescribed above may be performed on a number of hosts who are known tobe healthy, i.e. do not have a disorder of the prostate. An averagelevel of PSR polypeptide could then be determined which will act as astandard against which levels of PSR polypeptides can be measured forthe identification of atypical amounts of PSR polypeptides in vivo.

In accordance with another aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of SEQ ID No.2 or for the mature polypeptide encoded by the cDNA of the clonedeposited with the American Type Culture Collection (ATCC) at 108101University Boulevard, Manasass, Va. 20110-2209 U.S.A. as ATCC DepositNo. 75913 on Oct. 11, 1994.

The polynucleotide of this invention was discovered in a cDNA libraryderived from a human prostate. The PSR gene is primarily expressed inthe prostate and has not been found in any other human cDNA tissuelibraries screened by the inventors (see Table 1 below). PSR contains anopen reading frame encoding a protein of 316 amino acid residues.

TABLE 1 Identification of PSR as a Prostatic Specific Gene Normal StageB2 Stage C All other Prostate Cancer Cancer tissues PSA 4 7 14 0 PAP 131 34 0 PSR 0 3 7 0 Total Clones 4472 956 3397 275,261 Sequenced

As shown in Table 1, three prostatic cDNA libraries were constructed andlarge numbers of clones from these three libraries were sequenced. Theclones identified from these libraries were compared with a data basewhich contains 275,261 independent cDNA clone identifications obtainedfrom more than 300 human cDNA libraries other than human prostatic cDNAlibraries. As illustrated in the table, human prostatic Specific Antigen(PSA) was identified 4 times in the normal human prostate library, 7times in a stage B2 human prostate cancer library and 14 times in astage C human prostate cancer library. Human prostatic acid phosphatase(PAP) was identified 13 times in a normal human prostate library, oncein a stage B2 human prostate cancer library and 14 times in a stage Cprostate cancer library. The prostatic specific reductase of thisinvention was identified 3 times in the stage B2 human prostate cancerlibrary and 7 times in the stage C prostate cancer library, and mostnotably was not identified at all in the normal human prostate libraryor from libraries derived from non-prostate tissues, indicating itsimportance as a marker for prostate disorders.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in SEQ ID No. 2 or that of the deposited cloneor may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the samemature polypeptide as the DNA of SEQ ID No. 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of SEQ IDNo. 2 or for the mature polypeptide encoded by the deposited cDNA mayinclude: only the coding sequence for the mature polypeptide or thecoding sequence for the mature polypeptide (and optionally additionalcoding sequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideor which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofSEQ ID No. 2 or the polypeptide encoded by the cDNA of the depositedclone. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in SEQ ID No. 2 or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of SEQ ID No. 2 or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in SEQ ID No. 1 or of the coding sequence of the deposited clone.As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. An example ofa marker sequence is a hexa-histidine tag which may be supplied by avector, preferably a pQE-9 vector, which provides for purification ofthe mature polypeptide fused to the marker in the case of a bacterialhost, or, for example, the marker sequence may be a hemagglutinin (HA)tag when a mammalian host, e.g. COS-7 cells, is used. The HA tagcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length polynucleotide of the invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNA which have a high sequence similarity tothe full length polynucleotide of the invention. Probes of this typepreferably have at least 30 bases and may contain, for example, 50 ormore bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene of the invention including regulatory andpromotor regions, exons, and introns. An example of a screen comprisesisolating the coding region of the full length gene of the invention byusing the known DNA sequence to synthesize an oligonucleotide probe.Labeled oligonucleotides having a sequence complementary to that of thegene of the present invention are used to screen a library of humancDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides . As hereinused, the term “stringent conditions” means hybridization will occuronly if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which retain substantially the same biological function oractivity as the mature polypeptide encoded by the cDNA of SEQ ID No. 1or the deposited cDNA.

Alternatively, the polynucleotide may have at least 10 ten bases,generally at least 20 bases or 30 bases, and more preferably at least 50bases which hybridize to a polynucleotide of the present invention andwhich has an identity thereto, as hereinabove described, and which mayor may not retain activity. For example, such polynucleotides may beemployed as probes for the polynucleotide of SEQ ID NO:1, for example,for recovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 10 bases, generally at least 20 or 30 bases and preferably atleast 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The ATCC number referred to above is directed to a biological depositwith the ATCC, 12301 Parklawn Drive, Rockville, Md. 202852. Since thestrain referred to is being maintained under the term of the BudapestTreaty, it will be made available to a patent office signatory to theBudapest Treaty.

The present invention further relates to a PSR polypeptide which has thededuced amino acid sequence of SEQ ID No. 2 or which has the amino acidsequence encoded by the deposited cDNA, as well as fragments, analogsand derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of SEQ ID No. 2 or that encoded by the deposited cDNA, meansa polypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of SEQ ID No. 2 orthat encoded by the deposited cDNA may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol). Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least a 70% identity)to the polypeptide of SEQ ID NO:2 and more preferably at least a 90%similarity (more preferably at least a 90% identity) to the polypeptideof SEQ ID NO:2 and still more preferably at least a 95% similarity(still more preferably at least a 95% identity) to the polypeptide ofSEQ ID NO:2 and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the PSR genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to those ofordinarily skill in the art.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropliate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 and Sf9;animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The PSR polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

In accordance with another aspect of the present invention there areprovided assays which may be used to screen for therapeutics to inhibitPSR, since PSR is a reductase and may be necessary for the proliferationof the prostate cancer cells. The present invention discloses methodsfor selecting a therapeutic which forms a complex with PSR withsufficient affinity to prevent the biological action of PSR. The methodsinclude various assays, including competitive assays where the PSR isimmobilized to a support, and is contacted with a natural substrate forPSR and a labeled therapeutic either simultaneously or in eitherconsecutive order, and determining whether the therapeutic effectivelycompetes with the natural substrate in a manner sufficient to preventbinding of PSR to its substrate. In another embodiment, the naturalsubstrate is labeled and the therapeutic is unlabeled. In a furtherembodiment, the substrate is immobilized to a support, and is contactedwith both labeled PSR and a therapeutic (or unlabeled PSR and a labeledtherapeutic), and it is determined whether the amount of PSR bound tothe substrate is reduced in comparison to the assay without thetherapeutic added. The PSR may be labeled with the anti-PSR antibodiesof the subject invention.

In another example of such a screening assay, there is provided amammalian cell or membrane preparation expressing the PSR polypeptideincubated with elements which undergo simultaneous oxidation andreduction, for example hydrogen and oxygen which together form water,wherein the hydrogen could be labeled by radioactivity, e.g., tritium,in the presence of the compound to be screened under conditions favoringthe oxidation reduction reaction where hydrogen and oxygen form water.The ability of the compound to enhance or block this interaction couldthen be measured.

Potential antagonists to PSR including antibody, i.e., an anti-idiotypicantibody as described above, or in some cases, an oligonucleotide, whichbinds to the polypeptide.

Another potential PSR antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251:1360 (1991)),thereby preventing transcription and the production of PSR. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the PSR polypeptide(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of PSR.

Potential antagonists also include a small molecule which binds to andoccupies the active site of the polypeptide thereby making the activesite inaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

The antagonists may be employed to treat prostate cancer, since theyinhibit the function of PSR which is necessary for the viability of theprostate cancer cells. The antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as hereinafterdescribed.

Fragments of the full length PSR gene may be used as a hybridizationprobe for a cDNA library to isolate the full length PSR gene and toisolate other genes which have a high sequence similarity to the PSRgene or similar biological activity. Probes of this type can be, forexample, between 20 and 2000 base pairs. Preferably, however, the probeshave between 30 and 50 bases. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete PSR gene including regulatory andpromotor regions, exons, and introns. An example of a screen comprisesisolating the coding region of the PSR gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

The PSR polypeptides or agonists or antagonists may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, intra-anal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

The PSR polypeptides and agonists and antagonists which are polypeptidesmay also be employed in accordance with the present invention byexpression of such polypeptides in vivo, which is often referred to as“gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral vectors hereinabove mentioned maybe derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,human immunodeficiency virus, adenovirus, Myeloproliferative SarcomaVirus, and mammary tumor virus. In one embodiment, the retroviralplasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μg of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1

Determination of PSR Gene Transcription in Tissue Other Than Prostate

To assess the presence or absence of active transcription of PSR mRNA,approximately 6 ml of venous blood is obtained with a standardvenipuncture technique using heparinized tubes. Whole blood is mixedwith an equal volume of phosphate buffered saline, which is then layeredover 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrenetube. The gradient is centrifuged at 1800×g for 20 min at 5° C. Thelymphocyte and granulocyte layer (approximately 5 ml) is carefullyaspirated and rediluted up to 50 ml with phosphate-buffered saline in a50-ml tube, which is centrifuged again at 1800×g for 20 min. at 5° C.The supernatant is discarded and the pellet containing nucleated cellsis used for RNA extraction using the RNazole B method as described bythe manufacturer (Tel-Test Inc., Friendswood, Tex.).

Two oligonucleotide primers are employed to amplify the PSR nucleotidesequence present in the sample: the 5′ primer is 5′AAGAGATCCAGACCACGACAGG 3′ (SEQ ID No. 3) and the 3′ primer is 5′AAGGCACAGTGCAGCCTGGTCT 3′ (SEQ ID No. 4). The reverse transcriptasereaction and PCR amplification are performed sequentially withoutinterruption in a Perkin Elmer 9600 PCR machine (Emeryville, Calif.).Four hundred ng total RNA in 20 μg diethylpyrocarbonate-treated waterare placed in a 65° C. water bath for 5 min. and then quickly chilled onice immediately prior to the addition of PCR reagents. The 50-μl totalPCR volume consisted of 2.5 units Taq polymerase (Perkin-Elmer). 2 unitsavian myeloblastosis virus reverse transcriptase (Boehringer Mannheim,Indianapolis, Ind.); 200 μM each of dCTP, DATP, dGTP and dTTP (PerkinElmer); 18 pM each primer, 10 mM Tris-HCl; 50 mM KC1; and 2 mM MgCl₂(Perkin Elmer). PCR conditions are as follows: cycle 1 is 42° C. for 15min then 97° C. for 15 s (1 cycle); cycle 2 is 95° C. for 1 min. 60° C.for 1 min, and 72° C. for 30 s (15 cycles); cycle 3 is 95° C. for 1 min.60° C. for 1 min., and 72° C. for 1 min. (10 cycles); cycle 4 is 95° C.for 1 min., 60° C. for 1 min., and 72° C. for 2 min. (8 cycles); cycle 5is 72° C. for 15 min. (1 cycle); and the final cycle is a 4° C. holduntil sample is taken out of the machine. The 50-μl PCR products areconcentrated down to 10 μg with vacuum centrifugation, and the entiresample is then run on a thin 1.2% Tris-borate-EDTA agarose gelcontaining ethidium bromide. A 1.2 Kb band indicates that genomic DNA isamplified, which is not indicative of prostate cancer metastases.However, if the band is somewhat smaller, a 567 base pair product, theindication is that the PSR mRNA is amplified by PCR and cells, otherthan the prostate, are activley transcribing PSR protein and arecirculating in the blood, i.e. metastisizing. All specimens are analyzedat least twice to confirm a positive or negative outcome.

Verification of the nucleotide sequence of the PCR products is done bymicrosequencing. The PCR product is purified with a Qiagen PCR ProductPurification Kit (Qiagen, Chatsworth, Calif.) as described by themanufacturer. One μg of the PCR product undergoes PCR sequencing byusing the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer9600 PCR machine as described by Applied Biosystems (Foster, Calif.).The sequenced product is purified using Centri-Sep columns (PrincetonSeparations, Adelphia, N.J.) as described by the company. This productis then analyzed with an ABI model 373A DNA sequencing system (AppliedBiosystems) integrated with a Macintosh IIci computer.

EXAMPLE 2

Bacterial Expression and Purification of PSR

The DNA sequence encoding PSR, ATCC #75913, is initially amplified usingPCR oligonucleotide primers corresponding to the 5′ sequences of theprocessed protein (minus the signal peptide sequence) and the vectorsequences 3′ to the PSR gene. Additional nucleotides corresponding toPSR are added to the 5′ and 3′ sequences respectively. The 5oligonucleotide primer has the sequence 5′ GATCGATGTCGACCTGTCCAGTGGGGTGTGTAC (SEQ ID No. 5) 3′ contains a SalI restrictionenzyme site followed by 20 nucleotides of PSR coding sequence startingfrom amino acid 10. The 3′ sequence 5′ATCGATCTCTAGATTATGTTAGTCTATTGGGAGGCCC 3′ (SEQ ID No. 6) containscomplementary sequences to an XbaI site and is followed by 23nucleotides of PSR coding sequence. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311).pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with SalI and XbaI. The amplified sequences areligated into pQE-9 and are inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture is then used totransform E. coli strain m15/pREP4 available from Qiagen under thetrademark M15/rep 4 by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). M15/rep4 contains multiple copies of the plasmid pREP4, whichexpresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedPSR is purified from this solution by chromatography on a Nickel-Chelatecolumn under conditions that allow for tight binding by proteinscontaining the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). PSR (90% pure) is eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 3

Expression of Recombinant PSR in COS cells

The expression of plasmid, PSR HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire PSR precursor and a HA tag fused in frameto its 3′ end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to our target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding PSR, ATCC #75913, is constructed by PCR on theoriginal full-length PSR clone using two primers: the 5′ primer 5′GATCGAAGTCCTTCCTTCTGTATATGGCTG 3′ (SEQ ID No. 7) contains a HindIII sitefollowed by 19 nucleotides of PSR coding sequence starting from theinitiation codon; the 3′ sequence 5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATTGGGAGGCCCAGCAGGT3′ (SEQ ID No.8) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 18 nucleotides of the PSR coding sequence(not including the stop codon). Therefore, the PCR product contains aHindIII site, PSR coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith HindIII and XbaI restriction enzyme and ligated. The ligationmixture is transformed into E. coli strain SURE (Stratagene CloningSystems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantPSR, COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the PSR HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1%; NP-40(non-ionic detergent), 0.1% SDS, 0.5% DOC, 50 mM Tris,pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate andculture media are precipitated with a HA specific monoclonal antibody.Proteins precipitated are analyzed on 15% SDS-PAGE gels.

EXAMPLE 4

Expression pattern of PSR in human tissue

Northern blot analysis is carried out to examine the levels ofexpression of PSR in human tissues. Total cellular RNA samples areisolated with RNAzol™ B system (Biotecx Laboratories, Inc. Houston,Tex.). About 10 μg of total RNA isolated from each human tissuespecified is separated on 1% agarose gel and blotted onto a nylonfilter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold SpringHarbor Press, (1989)). The labeling reaction is done according to theStratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G-50 column. (5 Prime-3 Prime, Inc. Boulder,Colo.). The filter is then hybridized with radioactive labeled fulllength PSR gene at 1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDSovernight at 65° C. After wash twice at room temperature and twice at60° C. with 0.5×SSC, 0.1% SDS, the filter is then exposed at −70° C.overnight with an intensifying screen (See Table 1).

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primer$further includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified $EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

8 1086 BASE PAIRS NUCLEIC ACID SINGLE LINEAR cDNA 1 CCGGCAGAGATGGTTGAGCT CATGTTCCCG CTGTTGCTCC TCCTTCTGCC CTTCCTTCTG 60 TATATGGCTGCGCCCCAAAT CAGGAAAATG CTGTCCAGTG GGGTGTGTAC ATCAACTGTT 120 CAGCTTCCTGGGAAAGTAGT TGTGGTCACA GGAGCTAATA CAGGTATCGG GAAGGAGACA 180 GCCAAAGAGCTGGCTCAGAG AGGAGCTCGA GTATATTTAG CTTGCCGGGA TGTGGAAAAG 240 GGGGAATTGGTGGCCAAAGA GATCCAGACC ACGACAGGGA ACCAGCAGGT GTTGGTGCGG 300 AAACTGGACCTGTCTGATAC TAAGTCTATT CGAGCTTGGG CTAAGGGCTT CTTAGCTGAG 360 GAAAAGCACCTCCACGTTTG GATCAACAAT GCAGGAGTGA TGATGTGTCC GTACTCGAAG 420 ACAGCAGATGGCTTTGAGAT GCACATAGGA GTCAACCACT TGGGTCACTT CCTCCTAACC 480 CATCTGCTGCTAGAGAAACT AAAGGAATCA GCCCCATCAA GGATAGTAAA TGTGTCTTCC 540 CTCGCACATCACCTGGGAAG GATCCACTTC CATAACCTGC AGGGCGAGAA ATTCTACAAT 600 GCAGGCCTGGCCTACTGTCA CAGCAAGCTA GCCAACATCC TCTTCACCCA GGAACTGGCC 660 CGGAGACTAAAAGGCTCTGG CGTTACGACG TATTCTGTAC ACCCTGGCAC AGTCCAATCT 720 GAACTGGTTCGGCACTCATC TTTCATGAGA TGGATGTGGT GGCTTTTCTC CTTTTTCATC 780 AAGACTCCTCAGCAGGGAGC CCAGACCAGG CTGCACTGTG CCTTAACAGA AGGTCTTGAG 840 ATTCTAAGTGGGAATCATTT CAGTGACTGT CATGTGGCAT GGGTCTCTGC CCAAGCTCGT 900 AATGAGACTATAGCAAGGCG GCTGTGGGAC GTCATTGTGA CCTGCTGGGC CTCCCAATAG 960 ACTAACAGGCAGTGCCAGTT GGACCCAAGA GAAGACTGCA GCAGACTACA CAGTACTTCT 1020 TGTCAAAATGATTCTCCTTC AAGGTTTTCA AAACCTTTAG CACAAAGAGA GCAAAACCTT 1080 CCAGCC 1086316 AMINO ACIDS AMINO ACID LINEAR PROTEIN 2 Met Val Glu Leu Met Phe ProLeu Leu Leu Leu Leu Leu Pro Phe 5 10 15 Leu Leu Tyr Met Ala Ala Pro GlnIle Arg Lys Met Leu Ser Ser 20 25 30 Gly Val Cys Thr Ser Thr Val Gln LeuPro Gly Lys Val Val Val 35 40 45 Val Thr Gly Ala Asn Thr Gly Ile Gly LysGlu Thr Ala Lys Glu 50 55 60 Leu Ala Gln Arg Gly Ala Arg Val Tyr Leu AlaCys Arg Asp Val 65 70 75 Glu Lys Gly Glu Leu VAl Ala Lys Glu Ile Gln ThrThr Thr Gly 80 85 90 Asn Gln Gln Val Leu Val Arg Lys Leu Asp Leu Ser AspThr Lys 95 100 105 Ser Ile Arg Ala Trp Ala Lys Gly Phe Lys Ala Glu GluLys His 110 115 120 Leu His Val Trp Ile Asn Asn Ala Gly Val Met Met CysPro Tyr 125 130 135 Ser Lys Thr Ala Asp Gly Phe Glu Met His Ile Gly ValAsn His 140 145 150 Leu Gly His Phe Leu Leu Thr His Leu Leu Leu Glu LysLeu Lys 155 160 165 Glu Ser Ala Pro Ser Arg Ile Val Asn Val Ser Ser LeuAla His 170 175 180 His Leu Gly Arg Ile His Phe His Asn Leu Gln Gly GluLys Phe 185 190 195 Tyr Asn Ala Gly Leu Ala Tyr Cys His Ser Lys Leu AlaAsn Ile 200 205 210 Leu Phe Thr Gln Glu Leu Ala Arg Arg Leu Lys Gly SerGly Val 215 220 225 Thr Thr Tyr Ser Val His Pro Gly Thr Val Gln Ser GluLeu Val 230 235 240 Arg His Ser Ser Phe Met Arg Trp Met Trp Trp Leu PheSer Phe 245 250 255 Phe Ile Lys Thr Pro Gln Gln Gly Ala Gln Thr Arg LeuHis Cys 260 265 270 Ala Leu Thr Glu Gly Leu Glu Ile Leu Ser Gly Asn HisPhe Ser 275 280 285 Asp Cys His Val Ala Trp Val Ser Ala Gln Ala Arg AsnGlu Thr 290 295 300 Ile Ala Arg Arg Leu Trp Asp Val Ile Val Thr Cys TrpAla Ser 305 310 315 Gln 22 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 3 AAGAGATCCA GACCACGACA GG 22 22 BASE PAIRS NUCLEIC ACIDSINGLE LINEAR Oligonucleotide 4 AAGGCACAGT GCAGCCTGGT CT 22 33 BASEPAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 5 GATCGATGTC GACCTGTCCAGTGGGGTGTG TAC 33 37 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide 6 ATCGATCTCT AGATTATGTT AGTCTATTGG GAGGCCC 37 30 BASEPAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 7 GATCGAAGTC CTTCCTTCTGTATATGGCTG 30 57 BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotide 8CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAT TGGGAGGCCC AGCAGGT 57

What is claimed is:
 1. An antibody or portion thereof capable ofspecifically binding to a protein consisting of the amino acid sequenceof SEQ ID NO:2.
 2. The antibody or portion thereof of claim 1 which is amonoclonal antibody.
 3. The antibody or portion thereof of claim 1 whichis a polyclonal antibody.
 4. The antibody or portion thereof of claim 1which is a chimeric antibody.
 5. The antibody or portion thereof ofclaim 1 which is a humanized antibody.
 6. The antibody or portionthereof of claim 1 which is an Fab fragment.
 7. The antibody or portionthereof of claim 1 which is a single chain antibody.
 8. The antibody orportion thereof of claim 1 which is immobilized.
 9. The antibody orportion thereof of claim 1 which is labeled.
 10. A compositioncomprising the antibody or portion thereof of claim 1 and apharmaceutically acceptable carrier.
 11. The composition of claim 10,wherein the antibody or portion thereof is a monoclonal antibody. 12.The composition of claim 10, wherein the antibody or portion thereof ishumanized.
 13. The composition of claim 10, wherein the carrier issaline.
 14. The composition of claim 10, wherein the carrier isdextrose.
 15. The composition of claim 10, wherein the carrier is water.16. The composition of claim 10, wherein the carrier is glycerol.
 17. Ahybridoma cell line that produces the monoclonal antibody of claim 2.18. The hybridoma cell line of claim 17 wherein the antibody or portionthereof is humanized.
 19. An antibody or portion thereof capable ofspecifically binding to a protein consisting of the amino acid sequencethat is encoded by the cDNA contained in ATCC Deposit No.
 75913. 20. Theantibody or portion thereof of claim 19 which is a monoclonal antibody.21. The antibody or portion thereof of claim 19 which is a polyclonalantibody.
 22. The antibody or portion thereof of claim 19 which is achimeric antibody.
 23. The antibody or portion thereof of claim 19 whichis a humanized antibody.
 24. The antibody or portion thereof of claim 19which is an Fab fragment.
 25. The antibody or portion thereof of claim19 which is a single chain antibody.
 26. The antibody or portion thereofof claim 19 which is immobilized.
 27. The antibody or portion thereof ofclaim 19 which is labeled.
 28. A composition comprising the antibody orportion thereof of claim 19 and a pharmaceutically acceptable carrier.29. The composition of claim 28, wherein the antibody or portion thereofis a monoclonal antibody.
 30. The composition of claim 29, wherein theantibody or portion thereof is humanized.
 31. The composition of claim28, wherein the carrier is saline.
 32. The composition of claim 28,wherein the carrier is dextrose.
 33. The composition of claim 28,wherein the carrier is water.
 34. The composition of claim 28, whereinthe carrier is glycerol.
 35. A hybridoma cell line that produces themonoclonal antibody of claim
 20. 36. The hybridoma cell line of claim 35wherein the antibody or portion thereof is humanized.